ABSTRACT
The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.
Subject(s)
Animals , Microfilament Proteins/isolation & purification , Plasmodium falciparum/chemistry , Actins/immunology , Actins/isolation & purification , Chromatography, Affinity/methods , Immunoblotting , Microfilament Proteins/immunology , Myosins/immunology , Myosins/isolation & purification , Precipitin TestsABSTRACT
Effects of uterine stretching and physiological hypertrophy on myosin isozyme were investigated in rat during pregnancy. Both nonpregnant and pregnant rat uteri express a single myosin band on native gels. Analysis of native myosin under denaturing conditions revealed two myosin heavy chains (MHCs) with molecular mass of 204 and 200 kDa respectively. Filamin, a 240 kDa protein co-electrophoreses with myosin on native gels. No correlation is found between regulatory myosin light chain phosphorylation and pattern of myosin isozymes or the MHC. The results suggest that uterine stretching and physiological hypertrophy during pregnancy do not induce any changes in uterine myosin isozyme.
Subject(s)
Animals , Female , Muscle, Smooth/enzymology , Myosins/isolation & purification , Pregnancy , Pregnancy, Animal/metabolism , Rats , Rats, Wistar , Uterus/enzymologyABSTRACT
A miosina de pulmao bovino foi extraida e purificada atraves de algumas modificacoes no metodo proposto por Murakami e Col. (Murakami, U. et al 1974) que inclui: a) Lavagem do tecido muscular; b) Extracao da miosina com solucao tampao contendo 0,3 KCl; c) Precipitacao da miosina por dialise; d) Dissolucao e dissociacao da miosina de actina em solucao tampao contendo 0,5M KCl, ATP e ions Mg2+; e) Ultracentrifugacao 110.000g/2-3 horas e f)Fracionamento com sulfato de amonio 45-55 por cento de saturacao. Com este procedimento a miosina foi purificada de 11,33 e 2,23 vezes, respectivamente, calculados com as atividades Ca2+ e K+, EDTA-ATPase. A miosina de pulmao bovino e composta de pelo menos tres tipos distintos de cadeias polipepitidicas, cujos pesos moleculares, estimados por eletroforese de gel de poliacrilamida 7,5 por cento (SDS-PAGE) foi 200.000, 19.000 e 16.000 daltons. A estrutura dessa molecula e classificada como do tipo II de acordo com a classificacao de Cheney & Mooseker. O perfil de aminoacidos foi, tambem avaliado mostrando composicao semelhantes a miosina de outras fontes. Avaliou-se o efeito da temperatura, pH, concentracao de KCl e ureia na atividade Ca2+ e K+-EDTA-ATPase. A miosina de pulmao bovino mostrou fenomeno bifasico na determinacao da energia de ativacao na presenca de Ca2+. O valor de Km para atividade Ca2+ ATPase foi determinado